Oral Abstract Session 01 - Global HIV Vaccine Enterprise

ORAL ABSTRACT SESSIONS
74
Oral Abstract Sessions
Oral Abstract Session 07: B Cell Responses
OA07.07
454 Pyrosequencing and Bioinformatics Analysis of
the Lineage of the Broad and Potent gp41-Directed
Antibody 10e8
G. Ofek 2 , J. Zhu* 2 , Y. Yang 2 , B. Zhang 2 , K. Mckee 2 , M. Louder 2 ,
J. Huang 1 , L. Laub 1 , M. Connors 1 , J. Mascola 2 , P.D. Kwong 2
1 Laboratory of Immunoregulation, NIAID, NIH, USA; 2 Vaccine
Research Center, NIAID, NIH, Bethesda, MD, USA
Background: The 10e8 antibody is a novel MPER-specific
antibody that is both broad and potent, and lacks detectable
autoreactivity. 454 pyrosequencing has recently been shown
to allow for a bioinformatics analysis of the genetic record of a
broadly neutralizing antibody, once its sequence is known.
Methods: 454 pyrosequencing of B cells from donor N152,
from whom 10e8 was isolated, was undertaken to characterize
additional variants of 10e8 and to define its lineage. PCR reactions
using 10e8 germline-specific heavy and light chain primers were
performed and 454 pyrosequencing results analyzed using a
multi-step in-house bioinformatics pipeline.
Results: 37,669 IGHV3-15 heavy chain sequences and 91,951
IGLV3-19 light chain sequences were obtained and the
distribution of these sequences analyzed by two key parameters
– divergence from germline and sequence identity to 10e8.
Grid-based clustering allowed for the selection of 61 heavy
chain sequences and 48 light chain sequences. These were
reconstituted with the appropriate 10e8 light or heavy chain
partner, expressed and tested for MPER recognition. 12 heavy
chain variants and 27 light chain variants were found to bind
MPER, and several of the reconstituted antibodies neutralized a
panel of HIV-1 isolates better than the original 10e8. Phylogenetic
analyses identified clonally-related sequences, and at least three
subgroups of 10e8-like heavy chains were observed. 10e8 light
chains meanwhile also showed a few subgroups, though with
less sequence variation.
Conclusion: 454 pyrosequencing was thus able to identify clonal
variants of 10e8 with substantially improved neutralization
potency. Corresponding phylogenetic analyses are providing
insight into the ontogeny of 10e8, and structural and biophysical
studies are underway to functionally characterize this lineage.
*These authors contributed equally to this work.
AIDS Vaccine 2012
OA07.08 LB
Engineered Mice and B Cell Lines Expressing Broadly
Neutralizing Antibodies and Their Unmutated
Precursors: Tools for HIV Vaccinology
D. Nemazee 1 , C. Doyle-Cooper 1 , T. Ota 1 , A.B. Cooper 1 ,
M. Huber 1 , E. Falkowska 1 , K. . Doores 1 , L. Hangartner 1 ,
K. Le 1 , D. Sok 1 , J. Jardine 1 , J. Lifson 2 , X. Wu 3 , J.R. Mascola 3 ,
P. Poignard 1 , J.M. Binley 4 , B.K. Chakrabarti 5 , W.R. Schief 5 ,
R.T. Wyatt 5 , D.R. Burton 5
1 The Scripps Research Institute, La Jolla, CA, USA; 2 Frederick
National Laboratory for Cancer Research, Frederick, MD, USA;
3 Vaccine Research Center NIAID, Bethesda, MD, USA; 4 Torrey
Pines Institute, La Jolla, CA, USA; 5 IAVI Neutralizing Antibody
Center, USA
Background: Eliciting broadly neutralizing antibodies (bNAbs) to
HIV Env through immunization has been problematic.
Methods: To better understand the requirements for activation
of B cells producing bNAbs, we generated a series of cell lines
and transgenic mice expressing such antibodies or selected
germline-reverted versions as B cell surface receptors. The
bNAb cell lines included those that recognize the CD4 binding
site (b12, VRC01, PGV04, PGV19, NIH45-46), the MPER of gp41
(4E10), and additional glycan-dependent sites on the trimer
(PG9, PG16, PGT145, 2G12, PGT128, PGT135, PGT121). Different
Env-containing antigens and virions were tested for the ability to
stimulate bNAb cell lines.
Mouse strains expressing germline or mutated forms of 4E10
and b12 bNAb Ig genes were generated by gene targeting to the
physiological loci. These “knock-in” mice were studied for their B
cell development, and responses to HIV immunogens.
Results: Many HIV Env antigen preparations, notably including
infection-competent pseudovirions, were poorly recognized by
high affinity bNAb-expressing cells, as measured by calcium flux
assay. However, other antigen forms were highly stimulatory:
in particular, soluble gp140 foldon trimers and a multimerized,
scaffolded epitope protein.
4E10, but not b12 knock-in mice showed signs of abortive B cell
development. b12 H mice had gp120-binding cells and responded
well in vivo to gp140 trimers.
Conclusion: Analysis of bNAb cell line activation suggested that
HIV is difficult to recognize by B cells, probably because of the
low density of surface proteins. Based on these results, soluble
gp140 trimers or epitope scaffolds might offer more promise
as vaccine candidates. In knock-in mice, primary 4E10 B cell
precursors appeared to be negatively selected, whereas b12 B
cells were normal and readily stimulated with gp140 trimers.